RESEARCH ARTICLE
◥STRUCTURAL BIOLOGY
Structures and gating mechanism
of human TRPM2
Longfei Wang1,2,Tian-MinFu1,2†,YimingZhou3,4, Shiyu Xia1,2,AnnaGreka3,4,HaoWu1,2†
Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated
with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain
responsible for binding adenosine diphosphate (ADP)–ribose (ADPR), and both ADPR and
calcium (Ca2+) are required for TRPM2 activation. Here we report cryo–electron
microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca2+.
NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM
homology region (MHR1/2/3) in the apo state but undergoes conformational changes
upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit
interaction. The binding of Ca2+further engages transmembrane helices and the conserved
TRP helix to cause conformational changes at the MHR arm and the lower gating pore
to potentiate channel opening. These findings explain the molecular mechanism of
concerted TRPM2 gating by ADPR and Ca2+and provide insights into the gating
mechanism of other TRP channels.
T
he transient receptor potential melastatin
(TRPM) family belongs to the superfamily
of transient receptor potential (TRP) ion
channels and is involved in multiple bio-
logical processes and diseases ( 1 – 3 ). In
humans, there are eight TRPMs, which share
sequence similarity but respond to different
stimuli ( 1 – 3 ). TRPM family members share a
core architecture that includes a large TRPM
homology region (MHR1 to 4), a six-helix trans-
membrane (TM) domain, a conserved TRP helix
region, a rib helix, and a pole helix (Fig. 1A). In
addition to the core, some TRPMs have an ad-
ditional enzyme domain that regulates gating
and are sometimes classified as chanzymes ( 1 ).
For example, TRPM2 has a C-terminal NUDT9
homology (NUDT9H) domain, which has a pre-
dicted fold belonging to the Nudix hydrolase
family that catalyzes the conversion of adenosine
diphosphate (ADP)–ribose (ADPR) to adenosine
monophosphate (AMP) and ribose-5-phosphate
(R5P). It has been debated whether the NUDT9H
domain of TRPM2 has enzymatic activity ( 4 , 5 ).
TRPM2 forms a Ca2+-permeable nonselective
cation channel gated by ADPR and Ca2+( 6 – 9 ).
ADPR is a metabolic product of nicotinamide
adenine dinucleotide (NAD) and accumulates
in cells upon oxidative stress. TRPM2 relays
oxidative stress to Ca2+signaling with many vital
physiological roles ( 10 – 13 ). In particular, TRPM2
plays prominent functions in immunity and in-
flammation, which include chemokine produc-
tion, inflammasome activation, and infection
control ( 8 , 14 – 18 ). Under pathological conditions
such as ischemia-reperfusion injury, inflamma-
tion, and Alzheimer’s disease, TRPM2 can be
activated by high levels of reactive oxygen spe-
cies through ADPR accumulation and exacer-
bates the diseases ( 19 , 20 ). Therefore, TRPM2 is
an attractive therapeutic target against chronic
inflammatory and neurodegenerative diseases.
Recently reported cryo–electron microscopy
(cryo-EM) structures of human TRPM4 ( 21 – 24 ),
Ficedula albicollisTRPM8 ( 25 ),Nematostella
vectensisTRPM2 (nvTRPM2) ( 26 ), and mouse
TRPM7 ( 27 ) have shed light on the closed, in-
active core architecture of TRPM family cation
channels. Here we determined the cryo-EM
structures of full-length human (Homo sapiens)
TRPM2 (hsTRPM2) in apo, ADPR-bound, and
ADPR- and Ca2+-bound states. Instead of being
flexibly linked to the C-terminal end as pre-
viously presumed ( 26 ), NUDT9H folds back
onto the N-terminal domain to form extensive
interactions with MHR through both intra- and
intersubunit contacts. Upon ADPR binding,
NUDT9H undergoes conformational changes
that trigger the rotation of MHR1/2 and dis-
lodging of the intersubunit interaction. This
ADPR-induced“priming”effect may further al-
low Ca2+binding to concertedly tilt the TRP
helix, twist the MHR, and rotate the gating S6
helix to open the channel. Unexpectedly, the
cryo-EM structure of zebrafish (Danio rerio)
TRPM2 (drTRPM2) in complex with ADPR and
Ca2+published recently shows ADPR binding
at the MHR1/2 region ( 28 ). Our further exper-
imental evidence confirmed the species-specificdifference in ADPR binding and gating using
NUDT9H forhsTRPM2 and MHR1/2 fordrTRPM2,
respectively.Structure of full-lengthhsTRPM2 in the
apo state
We expressedhsTRPM2 in human embryonic
kidney (HEK) 293F cells and purified it in the
absence of ADPR and Ca2+(fig. S1). We then de-
termined its fourfold symmetric cryo-EM struc-
ture at 3.6-Å resolution (figs. S2 and S3, and
table S1). The initial cryo-EM density map was
of sufficient quality for modeling the N-terminal
cytosolic domains but not NUDT9H and the
TM region. Using density subtraction and focused
three-dimensional (3D) classification followed
by local refinement, we improved the cryo-EM
maps at these regions (figs. S2 and S3). The
final map revealed a square-shaped structure
with approximate dimensions of 150 Å by 100 Å
by 100 Å, and the final model comprises all do-
mains except the linker between the pole helix
andtheNUDT9Hdomain(Fig.1,AandB).
The overall structure ofhsTRPM2 is similar to
that ofdrTRPM2 ( 28 ) (fig. S4A), with NUDT9H
prominently decorating the bottom corners of
the large intracellular region of the tetramer
(Fig. 1, B and C). In a three-tier description of
the overall architecture that has been used for
TRPM channels, NUDT9H resides on the bottom
tier, which also comprises the N-terminal MHR1/2,
MHR3, and the pole helix (Fig. 1, C to E). Sur-
roundedbyMHR1/2,thesymmetricpolehelices
form a parallel tetrameric coiled coil at the
fourfold axis. The middle tier of TRPM2 consists
of the rib helix and the MHR4 domain; the
latter provides stackedahelices to bridge the
N-terminal cytosolic domain and the TM region.
We call the entire MHR region the MHR arm
(Fig. 1A). The top tier is composed of pre-S1, S1
to S6 TM domain helices, and the TRP helices.
As in most classic six-pass cation channels, the
TM region of TRPM2 is arranged in a domain-
swapped architecture, where the S1-S4 voltage
sensing–like domain (VSLD) of one subunit in-
teracts with the S5-S6 pore domain of a neigh-
boring subunit (fig. S4, B and C).Cis and trans interactions between
NUDT9H and MHRs
NUDT9H adopts a two-domain architecture, in
which the N-terminal domain (NTD) is domi-
nated by loops and shortbstrands, and the
C-terminal domain (CTD) comprises a central
bsheet surrounded by severalahelices (Fig. 2A).
NUDT9H resembles human NUDT9 ( 29 ), a mito-
chondrial ADPR pyrophosphatase, and has a con-
served P loop that connects twobstrands in
the centralbsheet of the NUDT9H CTD (Fig.
2, A and B). NUDT9H lies adjacent to MHR1/2
and MHR3 in the same subunit and also in-
teracts with MHR1/2 of a neighboring subunit.
We defined three contact areas: interfaces I, II,
and III (Fig. 2C). Interfaces I and II mediate
intrasubunit (cis) interactions of NUDT9H with
MHR1/2 and MHR3, respectively (Fig. 2, D and
E). Interface III enables intersubunit (trans)RESEARCH
Wanget al.,Science 362 , eaav4809 (2018) 21 December 2018 1of7
(^1) Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02115,
USA.^2 Program in Cellular and Molecular Medicine, Boston
Children’s Hospital, Boston, MA 02115, USA.^3 Department of
Medicine, Brigham and Women’s Hospital and Harvard
Medical School, Boston, MA 02115, USA.^4 Broad Institute of
MIT and Harvard, Cambridge, MA 02142, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected]
(T.-M.F.); [email protected] (H.W.)
on December 20, 2018^
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